Factors Affecting Microbial Contamination of Market Eggs: A Review

Abstract

The aim of the review was to analyze the ways of microbial contamination, the protective mechanism of egg, and factors that affect the quantity of contamination and microbial penetration. Eggs can be contaminated during their formation in the infected reproductive organs of hens or after laying, when eggs are exposed to contaminated environment. The eggs are equipped against microbial contamination by several protective mechanisms comprising the presence of cuticle, eggshell, eggshell membranes, occurrence of some antibacterial proteins, and high pH value of albumen. There are several factors that affect the quantity of microbial contamination and penetration such as species of bacteria, the amount of microorganisms, storage conditions, quality of eggshell or number of pores.

Full text

226 Scientia agriculturae bohemica, 45, 2014 (4): 226–237
INTRODUCTION
The external and internal quality of eggs is influ-
enced by a broad range of factors. This is because egg
quality criteria include such diverse and important
aspects as safety, nutritional and organoleptic proper-
ties or technological properties, all of which must be
controlled from farm to fork. For the poultry breeder,
farmers, food, egg sorting, and marketing companies,
the main priorities are to deliver a safe product which
is accepted by the consumers (N y s , 2009).
Eggs contaminated by microorganisms play a sig-
nificant role in poultry production pathology and
in the spreading of diseases. Microorganisms cause
increased mortality of embryos, lower hatchability,
and increased early chick mortality. Infections of
humans are also common (M i l a k o v i c - N o v a k ,
P r u k n e r , 1990).
In early studies, bacterial eggshell contamination
has been compared in litter and wire floor housing.
Q u a r l e s et al. (1970) reported that litter housing
had on average 9 times more bacteria in the air, and
20–30 times more aerobic bacteria on the shell than
wire floor housing. H a r r y (1963) reported that the
shells of eggs from deep litter systems had 15 times
more bacteria and a higher proportion of potential
spoilage organisms than eggs from battery cage sys-
tems. Conventional cage housing for laying hens is
prohibited starting in 2012 in the European Union,
following Council Directive 1999/74/EC.
From 2012 onward, only furnished cages and noncage
systems (aviaries and floor housing) are allowed.
A greater attention was given to the effect of hous-
ing system on egg hygiene. The development toward
furnished cages and noncage systems may have conse-
quences for egg hygiene by increasing the percentage
of cracked and dirty eggs (Wa l l , T a u s o n , 2002)
or the bacterial eggshell contamination (D e R e u et
al., 2005a; M a l l e t et al., 2006).
F i k s - v a n N i e k e r k (2005) pointed out high
eggshell contamination in an alternative system as well
as a positive correlation between the total airborne bac-
teria count in the housing system and the initial eggshell
contamination, as reported by P r o t a i s et al. (2003).
FACTORS AFFECTING MICROBIAL CONTAMINATION
OF MARKET EGGS: A REVIEW*
J. Svobodová, E. Tůmová
Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural
Resources, Prague, Czech Republic
The aim of the review was to analyze the ways of microbial contamination, the protective mechanism of egg, and factors that
affect the quantity of contamination and microbial penetration. Eggs can be contaminated during their formation in the infect-
ed reproductive organs of hens or after laying, when eggs are exposed to contaminated environment. The eggs are equipped
against microbial contamination by several protective mechanisms comprising the presence of cuticle, eggshell, eggshell
membranes, occurrence of some antibacterial proteins, and high pH value of albumen. There are several factors that affect the
quantity of microbial contamination and penetration such as species of bacteria, the amount of microorganisms, storage con-
ditions, quality of eggshell or number of pores.
laying hen; egg quality; penetration of microorganisms
* Supported by the Ministry of Agriculture of the Czech Republic, Project No. QJ1310002 and by IGA (Internal Grant Agency of the
Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Science Prague), Project No. SV 13-64-21320).
doi: 10.1515/sab-2015-0003
Received for publication on December 12, 2013
Accepted for publication on April 11, 2014

Scientia agriculturae bohemica, 45, 2014 (4): 226–237 227
D e R e u et al. (2006b) reported the significantly
(P ˂ 0.001) higher average eggshell contamination
by aerobic bacteria in eggs coming from alternative
housing systems as compared to those coming from
conventional ones, in particular 5.46 against 5.08 log
colony forming units (cfu) per eggshell. D e R e u
et al. (2005b) found a positive correlation between
the concentration of bacteria in the air of the poultry
house and the initial eggshell contamination regarding
total aerobic count. This study also showed that floor
eggs have a high bacterial load compared to eggs laid
in nest and that the egg conveyor belt is a key point
for contamination of accumulated eggs. D e R e u et
al. (2006a) and M e s s e n s et al. (2007) proved that
higher eggshell contamination led to a greater pos-
sibility of microorganism penetration and egg content
contamination.
One of the benefits of conventional battery cages
is that birds are separated from their manure in a very
efficient way. In furnished cages the presence of perches
may impair bird’s ability to efficiently trample the ma-
nure down through the cage floor (A b r a h a m s s o n ,
T a u s o n , 1993). Furthermore, how perches, litter
areas, and nests are situated in relation to each other
has impact on the hygiene of cage environment and
eggs (M a l l e t et al., 2006). In a study of Wa l l et
al. (2008), the proportions of dirty eggs were 4.2 and
5.4% in furnished and conventional cages, respectively.
Their results and other recently published studies show
that with well-designed furnished cages it is possible
to achieve similar results regarding proportions of dirty
eggs as in conventional cages (M a l l e t et al., 2006;
Wa l l , T a u s o n , 2007). D e R e u et al. (2005a)
compared the bacterial eggshell contamination of eggs
laid in conventional cages with eggs laid in the nest
boxes of furnished cages. No systematic difference in
shell contamination with total counts of aerobic bac-
teria was found between these systems (ranging from
4.0–4.5 log cfu per eggshell). Also, for Gram-negative
bacteria no difference was detected (both means ca.
3.0 log cfu per eggshell). M a l l e t et al. (2006) also
analyzed visually clean eggs and found that eggs laid
in the nests of furnished cages had similar bacterial
counts as eggs produced in conventional cages. In their
study nests were only partly lined with artificial turf,
leaving the wire mesh floor bare in the front part of the
nest (G u e s d o n et al., 2006). M a l l e t et al. (2006)
studied the hygienic aspects of eggs laid at different
locations in furnished cages. A significant differences
in total count of aerobic bacteria was observed on
the eggshell of eggs collected from furnished cages
(4.83 log cfu per eggshell) compared to conventional
cages (4.56 log cfu per eggshell). Wa l l et al. (2008)
also found a higher bacterial load on eggs from furnished
cages compared to conventional cages. The bacterial
counts were signicantly (P ˂ 0.001) higher in the fur-
nished cages compared to the conventional cages as
regards Enterococcus and total number of aerobic bacteria.
In further experimental studies, it was found that
eggs from aviaries were contaminated with higher num-
bers of aerobic bacteria than eggs from cage systems
(P r o t a i s et al., 2003; D e R e u et al., 2005a). The
difference was more than 1 log unit (up to 5.1–6.0 log
cfu per eggshell for eggs from aviaries), with much
higher counts on those eggs laid on the floor of the
aviaries (up to 7 log cfu per eggshell). For Gram-
negative bacteria no systematic differences were found
between cage and non-cage housing systems (D e
R e u et al., 2005a). In the study of D e R e u et al.
(2009) considerable differences were found in eggshell
contamination with total count of aerobic bacteria,
both for furnished cages (range 4.24–5.22 log cfu
per eggshell) and noncage systems (range 4.35–5.51
log cfu per eggshell). On the other hand, within the
noncage systems, the average eggshell contamination
with total count of aerobic bacteria found in four-floor
aviary housing systems (5.00 log cfu per eggshell) was
not significantly different from the average contami-
nation in three-floor aviary systems (4.95 log cfu per
eggshell). H u n e a u - S a l a ü n et al. (2010) found in
their study that within each type of housing system
there was no difference of shell contamination between
free range and organic flocks. In the study of D e R e u
et al. (2007) content contamination was 1.9% (5 out
of 269 eggs) for furnished cages compared to 2.3%
(10 out of 432 eggs) for non-cage systems.
The bacterial contamination of eggshells is af-
fected by several factors such as the concentration of
bacteria in the air of the poultry house (D e R e u et
al., 2005a) or birds’ diet (S m i t h et al., 2000). Diets
increasing the moisture of birds’ diet excreta not only
lead to higher proportions of excreta-contaminated
eggs but also increase the microbial contamination of
ostensibly clean eggs (S m i t h et al., 2000).
In some studies the total count of bacteria in the
air of poultry houses was proven to be positively cor-
related with the initial bacterial eggshell contamination
at the henhouse (P r o t a i s et al., 2003; D e R e u et
al., 2005a). Averages of 4 log cfu per m3 air for the
conventional and furnished cages were found compared
with a 100 times higher average (˃ 6 log cfu per m3)
for aviary housing systems (P r o t a i s et al., 2003).
Aerial dust monitoring showed that the dust con-
centration was higher in on-floor hen houses than in
conventional cage poultry houses (H u n e a u - S a l a ü n
et al., 2010). Ta k a i et al. (1998), Ellen et al. (2000),
and G u i l l a m et al. (2007) also reported higher dust
concentrations in perchery and aviary systems than
in cage poultry houses. Because dust contains bacte-
ria (Lyngtveit, Eduard, 1997; Radon et al.,
2002), the airborne bacterial concentration in on-floor
premises is likely to be higher than in conventional
cage hen houses (P r o t a i s et al., 2003; D e R e u et
al., 2005a). This poor microbiological air quality in
alternative housing systems may affect the bacterial
concentration on the eggs (Q u a r l e s et al., 1970).

228 Scientia agriculturae bohemica, 45, 2014 (4): 226–237
H u n e a u - S a l a ü n et al. (2010) reported that main
factor influencing aerial dust concentration in on-floor
systems was the addition of straw or sand to the litter
area at the beginning of the laying period. Adding a
substrate for dust bathing in the litter area led to an
increase in aerial air dust on the eggs. D e R e u et
al. (2005a) found that the eggshell contamination
as well with total count of aerobic bacteria as with
Gram-negative bacteria significantly decreased during
the winter period (up to ˃ 0.5 log cfu per eggshell;
P ˂ 0.05). Ta k a i et al. (1998) also reported a seasonal
influence on the dust concentration in poultry houses.
Some results of Q u a r l e s et al. (1970) also suspected
that high temperatures might influence the degree of
bacterial contamination on the eggshell.
Vertical transmission of bacterial infection
There are two possible ways of bacterial infection of
egg shells, vertically or horizontally. The vertical trans-
mission occurs in the reproductive organs of infected
hens namely from infection of ovaries by systemic
infection or ascending infection from contaminated
cloaca into the vagina and lower regions of the oviduct
(K e l l e r et al., 1995; M i y a m o t o et al., 1997). In
the transovarian route (vertical transmission), the yolk
(very infrequently the yolk itself), the albumen, and
the membranes are directly contaminated as a result
of bacterial infection of the reproductive organs, i.e.
ovaries or oviduct tissue, before the eggs are covered
by the shell (M e s s e n s et al., 2005a). Horizontal
transmission occurs when eggs are subsequently ex-
posed to a contaminated environment and microor-
ganisms penetrate the eggshell. Studies conducted
by B a r r o w, L o w e l l (1991) suggest that most of
the contamination is due to horizontal transmission,
although others do not agree (H u m p h r e y , 1994).
For some bacterial species and serotypes, trans-
ovarian and oviducal contamination may be very im-
portant (B a r n h a r t et al., 1991; G a s t et al., 1992;
B a u m l e r et al., 2000; R i c k e et al., 2001). In this
way, the eggs may be contaminated with bacteria such
as Salmonella or Campylobacter. For most serotypes
of Salmonella, trans-shell contamination is probably
the most important route of egg contamination. In the
case of Salmonella Enteritidis, this does not appear
to be the case. Salmonella Enteritidis is recovered
from egg contents but not from shells or from hen
faecal samples. Many authors report that Salmonella
Enteritidis is the dominant serotype isolated from egg
contents (Paul, Batchelor 1988; Perales,
Audicana 1988; Humphrey 1989; Mawer et
al., 1989). The deposition of Salmonella inside eggs
is thus most likely a consequence of reproductive
tissue colonization in infected laying hens (K e l l e r
et al., 1995; M e t h n e r et al., 1995; G a s , H o l t ,
2000). C o x et al. (1999, 2000) published molecular
evidence of transmission of Campylobacter from hens
to progeny through the fertile eggs. Examination of
oviducts from broiler breeder hens revealed infrequent
contamination as high as the isthmus with segments
closer to the vent yielding a greater number of posi-
tive (B u h r et al., 2001). However C o x et al. (2004)
found stronger evidence than transovarian or oviducal
contamination of Campylobacter. Immature follicles
and mature follicles examined were found to be 11.6%
and 25.7% Campylobacter contaminated.
It is generally believed that colonization of the
reproductive organs is a consequence of systemic
spread of Salmonella from the intestine (Va z q u e z -
T o r r e s et al., 1999). Invasion in the intestinal epithe-
lial cells triggers infiltration of immune cells, mainly
macrophages, resulting in the uptake of bacteria by
these cells. Because of its capability to survive and
replicate in the immune cells, bacteria carried in the
macrophages are spread within the host, resulting in
colonization of the reproductive organs (K e l l e r et
al., 1995; M i y a m o t o et al., 1997; O k a m u r a et
al., 2001; G a s t et al., 2007; G a n t o i s et al., 2008).
A systemic Salmonella Enteritidis infection in
laying hens can lead to the colonization of the ovary
or the oviduct (Keller et al., 1995; M i y a m o t o et
al., 1997; O k a m u r a et al., 2001; D e B u c k et al.,
2004a). Both organs can be infected independently
from each other (K i n d e et al., 2000), at the same
time or maybe one after the other. The extensive per-
meability of the vascular endothelia observed in the
ovary may contribute to the high colonization rate at
this site (G r i f f i n et al., 1984). In the majority of
experimental studies in laying hens, a higher frequency
of ovary colonization is reported, compared with the
frequency of recovery from the oviduct (D e B u c k
et al., 2004b; G a n t o i s et al., 2006; G a s t et al.,
2007). Therefore, it is strongly believed that Salmonella
Enteritidis must interact with the cellular components
of the preovulatory follicles. It was indeed shown that
Salmonella Enteritidis can attach to developing and
mature follicular granulosa cells exhibiting different
attachment patterns (T h i a g a r a j a n et al., 1994).
Higher bacterial numbers in the membranes of the
preovulatory follicles than in the yolk itself suggest
that during transovarian transmission, Salmonella
Enteritidis remains attached to the egg vitelline mem-
branes. A previous study has also suggested that yolk
contamination is more often associated with the vitelline
membrane than with the interior yolk contents (G a s t ,
B e a r d , 1990; G a s t , H o l t , 2000). Despite the fact
that many authors reported the vitelline membrane as
the most common site of Salmonella contamination
(Bichler et al., 1996; Gast, Holt, 2000; Gast
et al., 2002), other reports point to albumen as the
principal site of contamination in eggs (S h i v a p r a s a d
et al., 1990; H u m p h r e y et al., 1991; K e l l e r et
al., 1995), indicating that Salmonella Enteritidis is
colonizing oviduct tissues. M i y a m o t o et al. (1997)
observed that developing eggs in a highly contami-

Scientia agriculturae bohemica, 45, 2014 (4): 226–237 229
nated oviduct are likely to be Salmonella positive.
Colonization of the reproductive tract can be the result
of an ascending infection from the cloaca (R e i b e r
et al., 1995; M i y a m o t o et al., 1997), a descend-
ing infection from the ovary (K e l l e r et al., 1995)
and/or systemic spread of Salmonella. Depending on
the site of contamination, i.e. the vagina, isthmus,
and magnum, Salmonella could be incorporated into
the eggshell, the eggshell membranes or the albumen.
Horizontal transmission of bacterial infection
The presence of many different bacterial species on
the surface of the shells of eggs represents a potential
risk of contamination of egg content. Surface contami-
nation however may be the result of either infection
of the lower reproductive tract or faecal contamina-
tion. The faecal contamination of eggs is improbable
to occur during oviposition in a healthy laying hen.
Naturally, when a healthy hen lays an egg, its bearing
everts the vagina beyond faecal alimentary tract. This
protects the emerging egg from faecal contamination.
In addition, the stretching of the cloacal lining effec-
tively makes the intestinal tract somewhat slit-like,
further reducing the opportunity for contamination of
eggshell. This fact explains why eggshells in healthy
hens are not soiled faeces at oviposition (D e B u c k
et al., 2004a). Albeit most eggs are microbiologically
sterile at the time of lay, opportunities for contamination
abound the instant they leave the oviduct (B o a r d ,
T r a n t e r , 1995). Egg temperatures are around 42°C,
generally warmer than ambient air. Eggs are infected
as they cool, creating a negative pressure that can pull
material into the pores. As a result, eggs are potentially
contaminated by any surface with which they come
into contact. Sources of bacterial shell contaminants
can include caging material, nesting materials, wa-
ter, hands, broken eggs, blood, insects, and transport
belting though dust, soil, and faeces are probably the
most important (B o a r d , T r a n t e r , 1995; R i c k e
et al., 2001; D a v i e s , B r e s l i n , 2003). The extent
of contamination is directly related to the cleanli-
ness of these surfaces (B o a r d , T r a n t e r , 1995).
S m e l t z e r et al. (1979) found that eggs laid on the
dirty chicken house floor were more likely to exhibit
internal bacterial contamination than were eggs laid in
a nest box. Also P a d r o n (1990) detected that when
eggs were placed on Salmonella-contaminated nest
box shavings for 10 min, the eggshell and membranes
were penetrated by Salmonella organism in 59% of
the samples.
Physical defence of eggs
The eggs have several protective elements which
can defend against microorganisms even when bacteria
penetrate through the eggshell and eggshell mem-
branes. Physical resistance to bacterial contamination
is provided by the cuticle, eggshell, inner eggshell
membrane, and the outer eggshell membrane (M a y e s ,
T a k e b a l l i , 1983; S o l o m o n , 1997). Sometimes
referred to as bloom or shell accessory material, the
cuticle is a 0.01 mm thick protein-like substance that
coasts the outside of the shell. Cuticle is deposited
onto the surface of eggs during the final 1-1.5 h prior
to oviposition (B a k e r , B a l c h , 1962). It provides
protection in two ways. First, by adding to shell thick-
ness, it increases shell strength. Secondly and most
importantly, it prevents flow of water, bacteria, or
other materials through the shell pores (M u s g r o v e ,
2004). Despite the fact that the cuticle allows gas
passage, it seems to effectively fill the pores of the
eggshell (B r u c e , D r y s d a l e , 1994). However,
this defence is not perfect. A small percentage of
eggs are laid without cuticle, these eggs may easily
be contaminated by water and carbon black (B o a r d ,
H a l l s , 1973). Normally, the cuticle is likely to be
under strong natural selection in birds such as peli-
cans or flamingos that live in damp and presumably
more microbiologically challenging environments
and that have much thicker cuticles than do chickens
or quail (K u s u d a et al., 2011). Even when cuticle
is present, for the first few minutes after lay it is an
ineffective barrier to bacterial invasion until it hardens
(S p a r k s , 1987). In recent studies (D e R e u et al.,
2006a; M e s s e n s et al., 2007), it was reported that
cuticle deposition is important for the prevention of
penetration, and in the absence of cuticle deposition,
penetration is a frequent event. However, some research
groups (N a s c i m e n t o et al., 1992; M e s s e n s et
al., 2005b) observed no correlation between cuticle
deposition and penetration of Salmonella through the
eggshell. In the study of B a i n et al. (2013) the pen-
etration of eggs by microorganisms has been shown
for the first time to be directly dependent on variation
in cuticle deposition within the natural range observed
within a flock of laying hens. Eggs with the poorest
cuticle deposition were most frequently penetrated,
whereas eggs with the best cuticle deposition were
never penetrated. Using a subjective assessment of the
eggshells’ staining characteristics, it was observed that
there is a great deal of variation in cuticle deposition
on eggs laid by individual hens and among different
breeds (B a l l et al., 1975; S p a r k s 1994). Further
evidence for breed differences were observations that
the cuticle is thicker in brown vs white eggs (S i m o n s ,
1971; B o a r d , H a l l s , 1973). Taken together, this
suggested that genetics may be responsible for a sig-
nificant part of this trait variation and there may be a
genetic link between pigment and cuticle deposition.
The eggshell is another and very important bar-
rier against the entry of microorganisms. Eggshell
formation occurs within the shell gland or uterus,
the next part of the oviduct and the part in which an
egg spends the greatest amount of time (ca. 20 h).
The shell is composed of calcium carbonate, organic

230 Scientia agriculturae bohemica, 45, 2014 (4): 226–237
compounds, magnesium carbonate, and phosphate.
Knob-like structures made in the mammilary layer
provide structure for calcium carbonate. Irregular
patterns of calcite crystals comprise the spongy layer.
Thousands of pores are formed throughout the spongy
layer (M u s g r o v e , 2004). Shell attains to 241–371 μm
in thickness (S o l o m o n , 1991).
The third effective barrier are eggshell membranes.
There are two shell membranes that are held closely
together except at the blunt end of the egg where the
air cell is located (R o m a n o f f , R o m a n o f f , 1949;
S o l o m o n , 1997). The inner membrane lies over
the albumen and the outer membrane is attached to
calciferous shell. The membranes are built up of three
distinct layers: the inner and outer membranes which
consist of a network of randomly oriented fibres and
a homogeneous third layer of electron-dense material
called the limiting membrane (B r u c e , D r y s d a l e ,
1994). This limiting membrane intermeshes with the
innermost region of the inner membrane fibres rather
than forming a separable and distinct layer (W o n g
L i o n g et al., 1997). Most researchers estimate the
outer membrane to be double the thickness of the in-
ner membrane with a combined thickness of approxi-
mately 80 μm. These membranes are thought to serve
as a bacterial filter (G a r i b a l d i , S t o k e s , 1958;
K r a f t et al., 1958). The time needed for bacteria
to penetrate the combined inner and outer eggshell
membranes is not clearly related to the amount of
open space between the fibres in the outer surface of
the outer membrane (B e r r a n g et al., 1999). When
comparing the shell, inner, and outer membranes for
ability to prevent bacterial entry, the inner membrane
is the most effective because of the tighter meshwork
of the inner membrane relative to the outer membrane
(L i f s h i t z et al., 1964).
In addition to these physical protective barriers
albumen also contributes to the mechanical protection
against microorganisms. With mechanical defence, it
is the viscosity of the proteins and the organization of
the albumen in the albuminous sac so that biological
structure is conferred on the egg. Viscosity hampers the
movement of bacteria that invade the shell membranes
so that they do not have an unimpeded passage to the
yolk. The albuminous sac of fresh eggs contributes to
the central location of the yolk, thus maintaining it at
the greatest distance from the contaminants restrained
by the shell membranes.
Chemical defence of eggs
In addition to their function as a physical bar-
rier, the eggshell and shell membranes also act as a
chemical barrier. Although antibacterial proteins have
been identified mainly in the albumen, proteins with
well-known antibacterial properties have also been
associated with the eggshell and shell membranes
(G a n t o i s et al., 2009). There are several impor-
tant naturally occurring antimicrobial compounds
within the albumen. Ovotransferrin and conalbumen
chelate metal ions, particularly iron (S t a d e l m a n ,
C o t t e r i l l , 1995). It has also been identified in the
shell membranes and the basal calcified layer, possibly
acting as a bacteriostatic filter (G a u t r o n et al., 2001).
Ovotransferrin appears to be the major contributor to
the egg’s defence against microbial infection and rot-
ting (S t a d e l m a n , C o t t e r i l l , 1995). By depriving
the microorganisms of Fe3+, ovotransferrin prevents
microbial multiplication over a temperature range of
0–35°C. Above this temperature, many organisms, in-
cluding strains of Escherichia coli, die as a consequence
of iron deprivation (Stadelman, Cotterill,
1995). Ovomucoid inhibits trypsin. Lysozyme causes
hydrolysis of β-1,4-glycosidic bonds in peptidoglycans
(M u s g r o v e , 2004). It is also abundant in the limiting
membrane and is also present in the shell membranes,
the matrix, and the cuticle of the eggshell (H i n c k e
et al., 2000). Ovoinhibitor inactivates several prote-
ases, ovoflavoprotein chelates riboflavin and avidin
binds biotin (Stadelman, Cotterill, 1995;
M u s g r o v e , 2004). Recently, ovocalyxin-36, a novel
chicken eggshell and eggshell membrane protein, has
been identified (G a u t r o n et al., 2006). This protein
is involved in antibacterial defence, and therefore it is
believed that ovocalyxin-36 is of paticular importance
to keep the eggs free from pathogens. Protein extracts
derived from the cuticle and the outer eggshell matrix
indeed possess antimicrobial properties against both
Gram-positive and Gram-negative bacteria (C o r d e i r o
et al., 20013).
In addition, albumen pH is in the alkaline range.
Immediately after oviposition, pH ranges 7.6–7.9,
but a gradual increase is observed during stor-
age. As carbon dioxide is lost to the environment,
pH increases to more than 9, beyond the tolerance of
most microorganisms. Lysozyme, conalbumen, and
pH are considered to be the most important of the
antimicrobial factors naturally occurring in albumen
(Mayes, Takeballi, 1983).
There are several factors that influence the extent
of microbial penetration into the egg. These factors
can be divided into external and internal. The external
factors include the species of bacteria, the amount
of microorganisms, method and storage conditions.
Species of bacteria contamining eggs
Gram-positive bacteria, probably because of their
tolerance of dry conditions, dominate the flora on
eggshells. In contrast, Gram-negative bacteria are the
principal contaminants of rotten eggs (S t a d e l m a n ,
C o t t e r i l l , 1995; D e R e u et al., 2006a). Rotten
eggs normally contain a mixed infection of Gram-
negative bacteria and on occasion, a few Gram-positive
organisms are present, too. The most common con-
taminants are the genera Micrococcus, Staphylococcus,

Scientia agriculturae bohemica, 45, 2014 (4): 226–237 231
Arthrobacter, Bacillus, Pseudomonas, Alcaligenes,
Flavobacterium, Escherichia, and Pseudomonas
(Stadelman, Cotterill, 1995). De Reu et
al. (2007) reported that the natural eggshell contami-
nation they found in their study was dominated by
Gram-positive Staphylococcus spp. (S. equorum subsp.
linens, S. equorum, S. lentus, and S. xylosus). Board,
T r a n t e r (1995) reported that because of their toler-
ance for dry conditions, the microflora of the eggshell
is dominated by Gram-positive bacteria which may
originate from dust, soil or faeces. They found that
Staphylococcus was also the most dominating microflo-
ra in the air of the poultry houses. As major egg content
contaminants of their study, Gram-negative bacteria
as Escherichia coli, Salmonella, and Alcaligenes sp.
and Gram-positive bacteria like Staphylococcus lentus,
Staphylococcus xylosus, and Bacillus sp. (D e R e u et
al., 2007) were found. M a y e s , T a k e b a l l i (1983)
and B o a r d , T r a n t e r (1995) found rotten eggs
normally contain a mixed infection of Gram-negative
and a few Gram-positive organisms. Some of the most
common spoilage types in their studies were members
of the genera Alcaligenes, Pseudomonas, Escherichia,
Proteus, and Aeromonas (Mayes, Takeballi,
1983). The results of the study by D e R e u et al.
(2006a) show the percentage of eggshell penetration
(agar approach) for all strains used, after 21 days
of incubation. Pseudomonas sp. and Alcaligenes sp.
followed by Salmonella Enteritidis penetrated most
frequently the eggshell. They accounted for 60, 58, and
43% of the agar-filled eggs penetration, respectively.
The contents of whole eggs were most frequently con-
taminated by Salmonella Enteritidis (33%) followed
by Carnobacterium sp. (17.5%).
There are large differences in the level of contami-
nation of eggshells. M e s s e n s et al. (2005b) and D e
R e u et al. (2006b) reported that increasing numbers
of microorganisms on the eggshell consequently in-
crease the risk of microbial eggshell penetration and
egg content contamination. Eggshell bacterial numbers
fluctuate widely, from zero to hundreds or even millions
(Mayes, Takeballi, 1983; Board, Tranter,
1995). The extent of contamination of hatching eggs
was reported by B o a r d , T r a n t e r (1995) with
a variation ranging from 102 up to 107 cfu for individual
eggshells. An average number of bacteria per shell is
considered to be, 100 000 for unwashed or untreated
eggs (B o a r d , 1966).
Method and conditions of eggs storage
From other external factors, that influence the size
of contamination and microbial penetration into the
egg, also the method and time of storage play a role.
Temperature is an important factor affecting the pen-
etration. Fast penetration is observed when a positive
temperature differential is created between the egg
(warm) and the bacterial suspension (cool) (M a y e s ,
Takeballi, 1983; Bruce, Drysdale, 1994).
It is believed that a positive temperature differential,
combined with the presence of moisture, provides
an ideal opportunity for the bacteria to penetrate the
eggshell (Bruce, Drysdale, 1994; Berrang
et al., 1999). Therefore, it is very risky, when eggs
are removed from refrigerated storage and placed at
room temperature, they may sweat due to condensa-
tion of water droplets on the egg surface (B r u c e ,
Drysdale, 1994).
The study of D e R e u et al. (2005b) on the influ-
ence of time, temperature, and atmospheric humidity
on the bacterial shell contamination showed that total
count of aerobic bacteria did not decrease statistically
significantly during the storage time of 14 days, neither
at room temperature and an atmospheric humidity of
50% (from 5.44 to 5.22 log cfu per eggshell) nor at
refrigerator temperature (5°C) and an atmospheric hu-
midity of 85% (from 5.44 to 5.33 log cfu per eggshell).
G e n t r y , Q u a r l e s (1972) reported no marked
differences in viable counts after 1-day storage of the
freshly laid eggs at 4°C. Contrary to the total count
of aerobic bacteria, the total count of Gram-negative
bacteria decreased statistically significantly at room
environment (from 4.04 to 3.23 log cfu per eggshell)
but not at refrigeration environment (from 4.04 to
3.66 log cfu per eggshell). This was probably due to
the lower humidity at room temperature.
D e R e u et al. (2007) in their study examined
the influence of storage time on the amount of con-
taminated eggs. After lay (day 0) the contamination
was 2.7% (15 out of 554 eggs) and 3.4% (18 out
of 532 eggs) after a 21-day storage. D e R e u et al.
(2006a) studied the influence of the storage time on the
penetration of various bacterial species. Independent
of the selected strain, the eggshell penetration was
observed most frequently at ca. day 4–5. At day 6
and day 14, respectively, up to 80% and more than
95% of the total eggshell penetration was observed.
The Salmonella Enteritidis upon storage at various
temperatures and relative humidity has been studied
by B r a u n et al. (1999). The level of Salmonella
Enteritidis penetration to the egg contents increased
with increasing temperature and relative humid-
ity. Recovery of Salmonella from the contents was
already observed by day 3 when eggs were stored
above 15°C. Storage temperature did however not
effect Salmonella Enteritidis penetration in another
study (Wa n g , S l a v i k , 1998). At 10°C, the first
penetration was observed after 15 days of storage. In a
study by R a d k o w s k i (2002), however, Salmonella
Enteritidis was not recovered from the egg contents up
to 21 days whatever the storage temperature (2–30°C)
and relative humidity (normal or elevated).
The internal factors affecting the likelihood of
bacterial penetration in eggs include the cuticles,
shells, and membranes. In the eggshell especially its
quality and porosity are important.

232 Scientia agriculturae bohemica, 45, 2014 (4): 226–237
Eggshell quality
The quality of eggshells is most commonly defined
in terms of the amount of shell present and is assessed
by measuring shell specific gravity, shell weight or
shell thickness (M e s s e n s et al., 2005a). Eggs with
low specific gravity, and hence thinner eggshells, were
more likely to be penetrated by Salmonella (Sauter,
P e t e r s e n , 1974) and Pseudomonas (Orel, 1959). No
effect of eggshell thickness on ability to penetrate was
however found by K r a f t et al. (1958), W i l l i a m s
et al. (1968), and S m e l t z e r et al. (1979). M e s s e n s
et al. (2005b) studied the influence of eggshell qual-
ity on penetration of Salmonella Enteritidis and they
found that the thickness of eggshell does not affect the
penetration of these bacteria. Similar results have been
achieved by D e R e u et al. (2006a), which compared
seven selected bacterial species. They concluded that
size of the eggshell or eggshell thickness had no sig-
nificant effect on penetration. Another vital considera-
tion is how well the eggshell is constructed and this
is where ultrastructural studies play an important role
(Roberts, Brackpool, 1994). Nascimento,
S o l o m o n (1997) reported that eggs judged visually
to have poorer quality eggshells were more likely to
allow Salmonella Enteritidis penetration. The great-
est variation in eggshell ultrastructure occurred in the
mammillary layer and various abnormalities have been
described. However N a s c i m e n t o et al. (1992) re-
ported that some of these abnormalities decreased while
others increased the resistance to bacterial penetration.
Many factors have been found to affect eggshell
quality: the age of the hen, the strain of bird, environ-
mental temperature, dietary factors, dietary electro-
lytes, stress, disease, and other chemical compounds
(Roberts, Brackpool, 1994).
The age of the hen is one of the important factors
affecting shell quality. The shell of the first and last
egg laid was reported to be thicker than that of eggs
in the middle of the clutch (M a y e s , T a k e b a l l i ,
1983). Bacterial contamination of air cells, shells, and
egg contents was more common in eggs from older
hens than from younger hens (J o n e s et al., 2002).
N a s c i m e n t o et al. (1992) reported an increas-
ing eggshell penetration from 12.9% (beginning of
lay) to 25% (end of lay) for Salmonella Enteritidis.
The results of D e R e u et al. (2006a) showed that
the bacterial eggshell penetration remained almost
constant during the entire laying period. At weeks
34, 46, 60, 69, and 74 average penetration percent-
ages for all selected strains together were respectively
30, 39, 41, 33, and 37%. The whole egg contamination
increased slightly with hen age from respectively 13,
13, and 15% in weeks 34, 46 and 60 to 26 and 20% in
weeks 69 and 74. Eggshell contamination increased
significantly with the age of the laying hens, both
in caged flocks and flocks kept in alternative sys-
tems (H u n e a u - S a l a ü n et al., 2010). According
to M a l l e t et al. (2003), contamination decreased
with the age of hens kept in conventional and in fur-
nished cages, but the authors attributed this decrease to
a seasonal effect. Wa l l et al. (2008) also found that
the age of hens did not affect the total count or the
presence of Enterococcus. On the other hand, a study
by Kretzshmar-McCluskey et al. (2009) found
that the microflora load on the shell increased as the
age of hens increased.
Genetic selection for higher egg production and
greater egg weight has tended to result in poorer quality
shells (R o b e r t s , B r a c k p o o l , 1994) which are
more prone to become contaminated, as demonstrated
by J o n e s et al. (2002). In his study there also differ-
ences between the strains were found. Control strain
consistently maintained a lower level of contamination
for both monitored organisms (Salmonella Enteritidis
and Pseudomonas fluorescens) in each sampling group.
The overall results of this study suggest that genetic
selection has altered the ability of eggs to resist mi-
crobial contamination and that screening for microbial
integrity should be considered in the selection process
among the laying egg breeders.
Eggshell porosity
The hen’s eggshell has numerous pores estimat-
ed to range from 7000–17 000 per egg (M a y e s ,
T a k e b a l l i , 1983) that are unbranched and capped
with organic material (the cuticle) (B o a r d , 1980).
Even in an egg having an undamaged cuticle, there
are at least 10–20 pores that lack either an adequate
cover or plug of cuticle. These uncovered, also termed
′patent′ pores, may provide the portals for bacteria to
infect the contents of the egg (K r a f t et al., 1958;
B o a r d , T r a n t e r, 1995). In addition, the cuticle in
older eggs becomes dehydrated, resulting in its shrink-
age, and the pores become more exposed to bacterial
penetration (M a y e s , Ta k e b a l l i , 1983). Current
evidence suggests that, while pores represent portals
of entry, their function as primary routes of transfer is
of secondary importance to the structural defects that
occur in many eggs, and that by virtue of their mag-
nitude, offer a much easier route (S o l o m o n , 1997).
Many of the pores are located around the equator or the
blunt end of the shell. Pore diameter ranges 9–35 µm.
These openings are wider at the top than at the bottom.
Some are malformed but many pores run from the outer
surface to the shell membrane (M u s g r o v e , 2004).
In the study of M e s s e n s et al. (2005b) bacterial
penetration was higher at the blunt pole of the egg
than at the apex. Out of all the penetrated eggshells
(155 in total), 72.9% were penetrated at the blunt pole
and 52% at the apex. Pore numbers were significantly
higher at the blunt pole, averaging 32 ± 22 pores per
cm2 than at the apex, averaging 26 ± 19 pores per cm2.
Similar results were also found by H a i g h , B e t t s
(1991), where penetration was higher at the blunt pole

Scientia agriculturae bohemica, 45, 2014 (4): 226–237 233
of the egg compared with that of the apex, as previ-
ously observed. It has been stated that this is due to
the greater porosity at the blunt pole. Although they
observed a higher porosity at the blunt pole, they did
not observe the influence of the number of pores on
eggshell penetration. D e R e u et al. (2006a) did not
find a correlation between the number of pores and the
bacterial eggshell penetration and between the loss of
weight at the pores and the whole egg contamination.
Fromm, Monroe (1960) and Board, Halls
(1973) correlated porosity with bacterial penetration,
R e i n k e , B a k e r (1966) refuted this view. The stud-
ies of N a s c i m e n t o et al. (1992) and H a r t u n g ,
S t a d e l m a n (1963) also supported that bacterial
eggshell penetration is not pore dependent. The fact that
some pores do not extend through the thickness of the
shell but end abruptly (S i l y n - R o b e r t s , 1983) and
cuticular capping and plugs often present on/into pores
preventing microbial penetration (B o a r d , H a l l s ,
1973), may contribute to these conflicting opinions.
CONCLUSION
The egg is most often contaminated by faeces,
soil, litter or equipment after laying. Eggs have an
impressive arsenal of antimicrobial protective mecha-
nisms. Size of bacterial contamination is influenced
by numerous factors, namely the bacterial species
and amount of bacteria, storage conditions, quality
of shell, and housing system. It was found that some
species of bacteria penetrate into the egg easier and
more often. The amount of microorganisms is one of
the most important factors. It was detected that penetra-
tion increases with a growing number of microorgan-
isms. High temperatures and humidity during storage
negatively affect the amount of microorganisms. The
eggshell quality is another important internal factor,
which is further affected by age, genotype or nutrition.
Especially older hens produce thinner shells, which
may result in a higher penetration of microorganisms
into the egg content. Genetic selection for higher egg
production and greater egg weight has tended to result
in poorer quality shells which are more prone to become
contaminated. Some studies have demonstrated that
the number of pores can affect the size of the penetra-
tion. A significant effect was observed especially in
the housing system of laying hens where the number
of microorganisms on the surface of eggshell is higher
in an alternative type of housing (aviary, litter or free
range) compared to cage systems.
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Corresponding Author:
Ing. Jana S v o b o d o v á , Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department
of Animal Husbandry, Kamýcká 129, 165 21 Prague 6-Suchdol, Czech Republic, phone: +420 224 383 060,
e-mail: janasvobodova@af.czu.cz